Labeling of proteomic samples during proteolysis for quantitation and sample multiplexing

Abstract

This invention related to methods useful in the labeling of multiple polypeptide samples and subsequent analysis of these samples by mass spectrometry, particularly in the high throughput proteomic setting.

1. A method for encoding a plurality of polypeptide samples for analysis by mass spectrometry, comprising, for each individual sample:
 (i) cleaving the amide backbone of polypeptides in said sample to form sub-populations of fragments having carboxy-terminal residues;  
 (ii) mass-modifying the carboxy-terminal residues of said fragments with one of at least two moieties of different molecular weight to produce a plurality of discrete populations of mass-modified fragments which differ in molecular weight by the addition of said moiety, wherein the moieties differ in molecular weight due to inclusion of isotypes of differing molecular weight,  
 
       wherein, for each individual sample, the mass-modification produces the plurality of various discrete populations labeled, in a predetermined ratio of said at least two moieties, and which ratio is different from one of said individual samples to the next amongst the plurality of polypeptide samples. 
     
     
       2. A method for encoding a plurality of polypeptide samples for analysis by mass spectrometry, comprising, for each individual sample:
 (i) cleaving the amide backbone of polypeptides in said sample to form sub-populations of fragments having carboxy-terminal lysine or arginine residues;  
 (ii) mass-modifying the carboxy-terminal residues of a first portion of said fragments with a first moiety, and mass-modifying the carboxy-terminal residues of a second portion of said fragments with a second moiety, wherein the two moieties have different molecular weights, and the ratio between the first and second portions of said fragments are predetermined;  
 (iii) combining the two portions produced in (ii) and producing a plurality of discrete populations of mass-modified fragments which differ in molecular weight by the difference in molecular weight between the first and second moieties,  
 
       wherein, for each individual sample, the mass-modification produces the various discrete populations reflecting said predetermined ratio, and which ratio is different from one of said individual samples to the next amongst the plurality of polypeptide samples. 
     
     
       3. The method of  claim 1 , wherein the moieties are selected from halide, phosphate, amine, alkyl, thiol, or hydroxyl moieties. 
     
     
       4. The method of  claim 1  or  3 , wherein the moieties are added by modification of a carboxyl group of said carboxy terminal residue. 
     
     
       5. The method of  claim 1 , wherein the moieties are added by modification of an amine group of said lysine or arginine residue. 
     
     
       6. The method of  claim 1 , wherein enzymatic digestion is used to cleave the amide backbone of the polypeptides. 
     
     
       7. The method of  claim 6 , wherein the enzymatic digestion includes treatment of the polypeptides with an enzyme which produces a carboxy terminal lysine and/or arginine residue, such as selected from the group of trypsin, Arg-C and Lys-C, or a combination thereof. 
     
     
       8. The method of  claim 1  or  6 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in the same reaction mixture. 
     
     
       9. The method of  claim 1  or  6 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in separate reaction mixture. 
     
     
       10. The method of  claim 1 , wherein said fragments are separated based on size, solubility, electric charge and/or ligand specificity prior to ionization. 
     
     
       11. The method of  claim 10 , wherein said fragments are separated using one or more procedures selected from the group of gel-filtration, isoelectric precipitation, electrophoresis, isoelectric focusing, ion exchange chromatography, and affinity chromatography. 
     
     
       12. The method of  claim 10 , wherein said fragments are separated using high performance liquid chromatography. 
     
     
       13. The method of  claim 1 , further comprising:
 (iii) analyzing the molecular weights of said fragments by mass spectrometry.  
 
     
     
       14. The method of  claim 1 , further comprising:
 (iii) ionizing said fragments to produce gas phase ions;  
 (iv) further fragmenting the gas phase ions under conditions which produce a population of daughter ions of incremental molecular weight, which population of daughter ions results substantially from fragmentation of the amide backbone of said fragments;  
 (v) determining the molecular weight of said gas phase ions and daughter ions by mass spectrometry; and  
 (vi) determining the sequence of at least a portion of the test peptide or test peptide or test polypeptide from the determined molecular weights.  
 
     
     
       15. The method of  claim 13  or  14 , wherein the mass spectrometry method used is selected from fast atomic bombardment (FAB), plasma desorption (PD), thermospray (TS), electrospray (ES) and matrix assisted laser desorption (MALDI). 
     
     
       16. The method of  claim 2 , wherein the moieties are selected from halide, phosphate, amine, alkyl, thiol, or hydroxyl moieties. 
     
     
       17. The method of  claim 2  or  16 , wherein the moieties are added by modification of a carboxyl group of said lysine or arginine residue. 
     
     
       18. The method of  claim 2  or  16 , wherein the moieties are added by modification of an amine group of said lysine or arginine residue. 
     
     
       19. The method of  claim 2 , wherein enzymatic digestion is used to cleave the amide backbone of the polypeptides. 
     
     
       20. The method of  claim 19 , wherein the enzymatic digestion includes treatment of the polypeptides with an enzyme selected from the group of trypsin, Arg-C and Lys-C, or a combination thereof. 
     
     
       21. The method of  claim 2  or  19 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in the same reaction mixture. 
     
     
       22. The method of  claim 2  or  19 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in separate reaction mixture. 
     
     
       23. The method of  claim 2 , wherein said fragments are separated based on size, solubility, electric charge and/or ligand specificity prior to ionization. 
     
     
       24. The method of  claim 23 , wherein said fragments are separated using one or more procedures selected from the group of gel-filtration, isoelectric precipitation, electrophoresis, isoelectric focusing, ion exchange chromatography, and affinity chromatography. 
     
     
       25. The method of  claim 23 , wherein said fragments are separated using high performance liquid chromatography. 
     
     
       26. The method of  claim 2 , further comprising:
 (iv) analyzing the molecular weights of said fragments by mass spectrometry.  
 
     
     
       27. The method of  claim 2 , further comprising:
 (iv) ionizing said fragments to produce gas phase ions;  
 (v) further fragmenting the gas phase ions under conditions which produce a population of daughter ions of incremental molecular weight, which population of daughter ions results substantially from fragmentation of the amide backbone of said fragments;  
 (vi) determining the molecular weight of said gas phase ions and daughter ions by mass spectrometry; and  
 (vii) determining the sequence of at least a portion of the test peptide or test peptide or test polypeptide from the determined molecular weights.  
 
     
     
       28. The method of  claim 26  or  27 , wherein the mass spectrometry method used is selected from fast atomic bombardment (FAB), plasma desorption (PD), thermospray (TS), electrospray (ES) and matrix assisted laser desorption (MALDI). 
     
     
       29. A method for quantitating the abundance of a given polypeptide present in a sample using mass spectrometry, comprising of:
 (i) cleaving the amide backbone of polypeptides in said sample to form sub-populations of fragments having carboxy-terminal lysine or arginine residues;  
 (ii) cleaving the amide backbone of a standard sample of said given polypeptide to form sub-populations of fragments having carboxy-terminal lysine or arginine residues;  
 (iii) mass-modifying the carboxy-terminal residues of fragments generated in step (i) with a first moiety, and mass-modifying the carboxy-terminal residues of fragments generated in step (ii) with a second moiety, wherein the two moieties have different molecular weights;  
 (iv) combining the two portions produced in (iii), and subjecting the peptide sample to analysis by mass spectrometry to generate mass spectra comprising at least one signal doublet for each fragment, the signal doublet comprising a first signal and a second signal shifted a known units from the first signal, wherein said known units is the difference in molecular weight between the two said moieties;  
 (iii) determining a signal ratio for at least one fragment pair by relating the difference in signal intensity or area between the first signal and the second signal;  
 
       whereby the abundance of the given polypeptide is determined from the said signal ratio and the known amount of said standard sample of the given polypeptide, based on the principle that signal intensity is proportional to peptide abundance. 
     
     
       30. The method of  claim 29 , wherein the first and second moieties used are different isotypes of the same atom. 
     
     
       31. The method of  claim 30 , wherein the isotypes used are  16 O and  18 O in H 2 O. 
     
     
       32. The method of  claim 29 , wherein enzymatic digestion is used to cleave the amide backbone of the polypeptides. 
     
     
       33. The method of  claim 32 , wherein the enzymatic digestion includes treatment of the polypeptides with an enzyme selected from the group of trypsin, Arg-C and Lys-C, or a combination thereof. 
     
     
       34. The method of  claim 29 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in the same reaction mixture. 
     
     
       35. The method of  claim 29 , wherein cleavage of the amide backbone of the polypeptides and the mass modification are carried out in separate reaction mixture. 
     
     
       36. The method of  claim 29 , wherein the mass spectrometry method used is selected from fast atomic bombardment (FAB), plasma desorption (PD), thermospray (TS), electrospray (ES) and matrix assisted laser desorption (MALDI). 
     
     
       37. The method of  claim 1 ,  2 , or  29 , wherein for each individual sample, a first protease is used to digest all polypeptides within said each individual sample before step (i). 
     
     
       38. The method of  claim 37 , wherein about 100 nL or less of all digested polypeptides from said each individual sample is adsorbed in a capillary and/or chromatographic materials therein, and a second protease is used to further cleave the amide backbone of the adsorbed polypeptides in step (i) at the presence of said first and/or said second moiety. 
     
     
       39. The method of  claim 38 , wherein the first protease is Lys-C, the second protease is trypsin. 
     
     
       40. The method of  claim 38 , wherein before adding the second protease, the adsorbed polypeptides are washed one or more times with a buffer. 
     
     
       41. The method of  claim 13 ,  14 ,  26 , or  27 , wherein pulsing is used to boost the signal of one or more of said fragments, or one or more of said gas phase ions and daughter ions.